The glycoprotein hormones lutropin (LH), follitropin (FSH), thyrotropin (TSH), and chorionic gonadotropin (CG) are a family of four closely related dimeric proteins which share common alpha-subunits but have hormone-specific beta-subunits. LH bears Asn-linked oligosaccharides which terminate with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1, 2Man alpha whereas FSH, synthesized in the same cell, contains Sialic acid-Gal in place of SO4-GalNAc. The goals of this proposal are to characterize in depth the two transferases we have identified whose sequential action is responsible for synthesis of these sulfated oligosaccharides, and to determine the biologic function of these sulfated structures. To accomplish this we will purify the sulfo- and GalNAc-transferases, and define their kinetic and substrate recognition parameters. cDNAs coding for the sulfo- and GalNAc-transferases will be prepared and sequenced to establish the primary structures of these membrane-bound transferases. We will determine if they have regions which are homologous to other glycosyltransferases and Golgi proteins, and may account for their specificities and localization within the Golgi. We have shown that some aspect of the peptide sequence in glycoprotein hormones is recognized by the GalNAc-transferase. The amino acid sequence of this recognition marker will be identified. The possible presence of this marker and sulfated structures on other types of glycoproteins will be assessed. We will explore whether the expression of these transferases is under hormonal regulation and results in the synthesis of oligosaccharides with altered structures. The functional significance of these oligosaccharides for sorting of LH and FSH to different granules within the gonadotroph, for regulating hormone bioactivity, and for determining clearance from the blood will be examined. The properties of glycoproteins in other cells bearing the same structures will be compared with those of the hormones to determine if their oligosaccharides have related functions. The glycoprotein hormones provide a model system for understanding how unique oligosaccharides arise on select families of proteins. Since only specific members of the glycoprotein hormone family bear sulfated oligosaccharides, this is an important model system which will allow us to develop a better understanding of the biologic significance of glycosylation.